The present invention relates to devices, systems and methods for genetic diagnostic applications, particularly to determine the presence or absence of Single Nucleotide Polymorphisms (SNP) within specific sequences of DNA.
The detection and screening of Single Nucleotide Polymorphisms (SNPs), is receiving increasing interest and effort in genomics research. SNPs are the most common type of DNA sequence variation and efforts are being made to generate sufficiently dense genetic maps for complex trait mapping. As a result, the number of SNP samples tested per year is increasing at a significant rate.
It is believed that SNPs are indicators to determine the pre-disposition of patients to diseases such as cancer, cardiovascular disease and other pathologies. SNPs also have application in pharmacogenetic applications and drug development, such as drug toxicity, metabolism, and efficacy. Further, SNPs have application for identifying bacterial mechanisms of antibiotic resistance. Scanning the human genome for sequence variations could identify millions of potentially informative genetic markers. These diagnostic applications require a large number of SNPs for definitive indications and should be compared against a large number of samples for accuracy.
Some of the sampling effort has been focused on oligo arrays, as well as other genetically based diagnostic applications. However, the present state of instrumentation, informatics and associated cost restrict the number of samples that can be run against these arrays.
It is an object of the present invention to provide devices, methods and systems for detection and screening of SNPs, particularly for detecting and screening SNPs on a faster and volumetric basis. It is also an object of the present invention to provide such apparatuses, methods and systems which are relatively inexpensive, easy-to-use and have flexibility or versatility in their uses.
It is a further object of the present invention to provide devices, systems and methods for detecting and screening of SNPs that make minimal use of custom automation and instrumentation. In this regard, it is desirable to utilize conventional instrumentation, such as fluid handling equipment and fluorescence readers.
It is still a further object of the present invention to provide devices, methods and systems for detecting and screening of SNPs that can screen large numbers of samples and at the same time minimize the required material volumes and resultant costs. It is an additional object of the present invention to provide a fluid sampling device with separate components and which can be disassembled, and which does not utilize separate gasket members or adhesives to hold and seal the components together.
In accordance with the present invention, devices, methods and systems are provided which perform genetic assays, particularly to determine the presence or absence of Single Nucleotide Polymorphisms (SNPs) within specific sequences of DNA. The inventive system basically comprises two main components, an analysis or assay device and a support base. The analysis device contains a housing, a multi-port middle application layer, and at least one glass slide member for specimens. The middle layer is made of a compliant, moldable, elastomer material with a plurality of channels or cavities molded into it. For example, the middle layer can be made from a polydimethylsiloxane (PDMS) material or a liquid silicone rubber (LSR) material, although the invention is not limited to these two materials. Each slide member contains spots or sites that comprise arrays of deposited oligonucleotides, each designed to detect a SNP of interest. The number of SNP tests per device depends on the design of the channels or cavities and the density of the array. The middle layer creates a tight liquid seal against the glass slide when the device is assembled. PDMS and LSR, in particular, have an affinity to stick tightly to glass and provide a reversible liquid tight seal. With the present invention, micro-sized channels and cavities can be formed within the self-sealing middle layer. Separate sealing members or adhesives are not needed to hold and seal the component members together.
Openings or ports are provided at opposite ends or surfaces of the analysis device, the ports being in liquid communication with the channels or cavities in the middle layer. The channels or cavities can be designed to address specific product requirements and preferably are very small microsized members. Also, due to the self-sealing characteristics of the middle layer, additional sealing devices or mechanisms are unnecessary at the ports and channels.
The middle layer and slide member(s) are positioned inside the housing. Two portions of the housing or frame member are snapped or otherwise held together forming the housing and holding the assembly together. Biasing members could also be provided if necessary to apply a constant slight pressure to the slide and middle member, if necessary, in order to improve the seal between them.
In use, appropriate liquid materials are introduced sequentially into the ports at one end or side of the analysis device in order to perform the assay or analysis intending to identify and/or detect the presence or absence of SNPs. Waste materials exit from ports in the opposite side of the device. Wash materials and reagents are circulated through the device as required.
Other embodiments of assay devices can also be utilized. A single sample device includes a cover-type housing in which a compliant, elastomer material and glass slide are positioned, the housing having only a single port for entry of DNA, reagents and other materials to form the SNPs from oligos spotted on the slide. An absorbent material can collect the waste materials which flow past the spots.
A plurality of assay devices can also be assembled together as a unit in a support base. A pumping mechanism or absorbent materials are preferably provided in the support base in order to remove the waste materials from the system. A group of twelve assay devices, each with eight ports form a microtiter arrangement in the support base and can be easily subjected to robotic or automated processing particularly with pressure pumping. In this regard, the present invention extends in the vertical direction of the volume of a microtiter plate and increases the usable surface area without increasing the horizontal area or footprint of a microtiter plate.
These and other features of the invention will become apparent from the following description of the invention, when viewed in accordance with the attached drawings and appended claims.